FIG. 5.

TFII-I is degraded after γ-irradiation in a p53- and ATM-dependent manner. (A) Abelson virus-transformed murine pre-B cells with either wild-type p53 or lacking p53 were irradiated at 10 Gy. Four hours post-gamma-irradiation treatment, whole-cell extracts were prepared and normalized by protein concentration. Lysates were analyzed by Western blotting. (B) The same procedures were followed as described for panel A except that the lysates were made 6 h after irradiation treatment. (C) NIH 3T3 cells with wild-type p53 were irradiated at 30 Gy. Eight hours post-gamma-irradiation treatment, whole-cell extracts were prepared and normalized by protein concentration. Lysates were analyzed by Western blotting. (D) COS cells were transiently transfected with GST-TFII-I and increasing amounts of HA-p53. Whole-cell extracts were prepared and normalized by protein concentration. Lysates were analyzed by Western blotting. (E) COS cells were transiently transfected with a cyclin D1-Luc reporter construct, p53, and increasing amounts of TFII-I. All concentrations are in nanograms, and experiments were repeated at least three times. (F) Wild-type p53, p53 null, p53 mutant, ATM null, and ATM null plus p53 mutant Abelson virus-transformed murine pre-B cells were irradiated at 10 Gy. Four hours post-gamma-irradiation treatment, whole-cell extracts were prepared and normalized by protein concentration. Lysates were analyzed by Western blotting. The asterisk denotes a nonspecific band used throughout as an additional loading control.